H. pylori was isolated in 1982 by B. Marshall and J. Warren using microaerophilic conditions that had been developed to grow Campylobacter jejuni. H. pylori bacteria are S-shaped, gram negative bacilli 2-3.5 μm in length and 0.5-1 μm in width (Blaser, M. J., and J. Parsonnet. 1994. J. Clin. Invest. 94:4-8). It is now (only recently) known that infection with H. pylori is the most common infection in the world. In developing countries 80% of the population is infected by the bacterium at the age of 20, while in developed countries H. pylori infection increases with age from <20% in 30-year old people to >50% in 60-year olds (Axon, A T. 1995. Pharmacol. Ther. 9:585-588; Blaser and Parsonnet, 1994). The infection is transmitted by either the oro-fecal or the oro-oral route (Blaser and Parsonnet, 1994). Infection occurs during the first years of life and persists forever. Once established, the infection is chronic, possibly permanent. Risk factors for infection are crowding, poor hygiene and host-specific genetic factors.
The complete genome sequence of H. pylori has been published (Tomb, J.-F., et al. 1997. Nature 388:539-547). A brief review of this article and a general review of the biology of H. pylori can be found in Doolittle, R. F. 1997. Nature 388:515-516.
Chronic infection of the human gastroduodenal mucosac by H. pylori is frequently associated with chronic gastritis, peptic ulcer, and increases the risk of occurrence of gastric malignancies such as adenocarcinoma and low grade B cell lymphoma (Blaser and Parsonnet, 1994; Parsonnet, J., et al. 1991. N. Engl. J. Med. 325: 1127-1231; Parsonnet, J., et al. 1994. N. Engl. J. Med. 330: 1267-1271). Most of the infections remain asymptomatic, whereas symptomatic, severe diseases correlate epidemiologically with the infection by a subset of H. pylori strains, called Type I (Blaser, M. J., et al. 1995. Cancer Res. 55:2111-2115; Covacci, A., et al. 1997. Trends Microbiol. 5:205-208; Covacci, A., et al. Proc. Natl. Acad. Sci. U.S.A. 90:5791-5795; Eck, M., et al. 1997. Gastroenterology. 112:1482-1486; Xiang, Z., et al. 1995. Infect. Immun. 63:94-98). This subset of strains is endowed with increased virulence due to the expression of a biologically active toxin (VacA), which is cytopathic to gastric epithelial cells in vitro and in vivo (Ghiara, P., et al. 1995. Infect. Immun. 63:4154-4160; Harris, P. R., et al. 1996. Infect. Immun. 64:4867-4871; Telford, J. L., et al. 1994. J. Exp. Med. 179:1653-1658), and also to the acquisition of a pathogenicity island (PAI), called cag, which contains a set of genes encoding several virulence factors (Censini, S., et al 1996. Proc. Natl. Acad. Sci. USA. 93:14648-14653) which are responsible for the induction of the synthesis of the neutrophil chemotactic cytokine IL-8 by the gastric epithelial cells (Censini et al, 1996).
H. pylori factors that have been identified so far include the flagella that are probably necessary to move across the mucus layer, see for example, Leying et al. 1992. Mol. Microbiol. 6:2863-2874; the urease that is necessary to neutralize the acid environment of the stomach and to allow initial colonization, see, for example, Cussac et al. 1992. J. Bacteriol. 174:2466-2473, Perez-Perez et al. 1992. J. Infect. Immun. 60:3658-3663, Austin, et al. 1992. J. Bacteriol. 174:7470-7473, WO 90/04030; the H. pylori cytotoxin (sometimes referred to as VacA, as it causes vacuolation), see, for example, WO 93/18150, Telford, J. L. et al. 1994. J. Exp. Med. 179:1653-1658, Cover et al. 1992. J. Bio. Chem. 267:10570-10575, Cover et al. 1992. J. Clin. Invest. 90:913-918, Leunk, 1991. Rev. Infect. Dis. 13:5686-5689; the H. pylori heat shock proteins (hsp), see, for example, WO 93/18150, Evans et al. 1992. Infect. Immun. 60:2125-2127, Dunn et al. 1992. Infect. Immun. 60:1946-1951, Austin et al., 1992; and the cytotoxin-associated protein, CagA, see, for example, WO 93/18150, Covacci, A., et al. 1993. Proc. Natl. Acad. Sci. USA 90:5791-5795, Tummuru, M. K., et al. 1994. Infect. Immun. 61:1799-1809.
Currently, H. pylori strains can be partitioned into at least two major groups, which either express (Type I) or do not express (Type II) the cytotoxin (VacA) and the CagA proteins. Type I strains contain the CagA and toxin genes and produce active forms of these antigens. Type II strains lack the CagA locus and fail to express the cytotoxin. The association between the presence of the CagA gene and cytotoxicity suggests that the production of the CagA gene is necessary for the transcription, folding, export or function of the cytotoxin. Epidemiological analysis indicate that Type I bacteria are associated with duodenal ulcerations, gastric ulceration and severe forms of active gastritis.
For a general review of the pathogenic role of H. pylori in peptic ulcer, see Telford, J. L., et al. 1994. TIBTECH 12:420-426.
H. pylori culture supernatants have been shown by different authors to contain an antigen with a molecular weight of 120, 128 or 130 kDa (Apel, et al. 1988. Zentralblat für Bakteriol. Microb. Und Hygiene 268:271-276; Crabtree, et al. 1992. J. Clin. Pathol. 45:733-734; Cover, et al. 1990. Infect. Immun. 58:603-610; Figura, et al. 1990. H. pylori, gastritis and peptic ulcer (eds. Malfrtheiner, et al.), Springer Verlag, Berlin). Whether the difference in size of the antigen described was due to interlaboratory differences in estimating the molecular weight of the same protein, to the size variability of the same antigen, or to actual different proteins was not clear. This protein is very immunogenic in infected humans because specific antibodies are detected in sera of virtually all patients infected with H. pylori (Gerstenecker, et al. 1992. Eur. J. Clin. Microbiol. 11:595-601).
A protein known as NAP (neutrophil activating protein—Evans D. J., et al. 1995. Gene 153:123-127; WO 96/01272 & WO 96/01273, especially SEQ ID NO:6; see also WO 97/25429), which is found in both type I and II strains, appears to be protective when tested in the H. pylori mouse model (Marchetti, M., et al. 1995. Science. 267:1655-1658). NAP is a homodecamer of 15 kDa subunits, and it has been proposed that the multimeric complex has a ring-shaped structure which spontaneously forms hexagonal paracrystalline structures. The assembled protein appears to interact with glycosphingolipid receptors of human neutrophils.
A number of other H. pylori antigens are described in WO 98/04702, including ureaseB (SEQ ID NO: 4), HopX (SEQ ID NO: 21), HopY (SEQ ID NO: 21), 36 kDa (SEQ ID NO: 26), 42 kDa (SEQ ID NO: 25), and 17 kDa (SEQ ID NO: 27). Urease is also described in, for example, EP-B-0367644 (protein with urease activity), EP-A-0610322 (ureaseE, F, G, H and I), EP-A0625053 (urease protein) and EP-A-0831892 (multimeric forms of urease). Other H. pylori antigens include the 54 kDa (SEQ ID NO: 2) and 50 kDa (SEQ ID NO: 1) proteins described in EP-A-0793676.
Discussions of various virulence factors of H. pylori can be found in, for example, EP-A-93905285.8 and EP-A-96908300.5.
Colonization of the mucosa of the stomach by H. pylori is today recognized as the major cause of acute and chronic gastroduodenal pathologies in humans (Blaser and Parsonnet, 1994; Covacci et al, 1997). The recognition of the infectious nature of the illness is having a major impact in the treatment of the disease that is shifting from the treatment of symptoms by anti-H2 blockers to the eradication of the bacterial infection by antibiotic regimen.
In spite of the unquestionable successes that will be achieved with antibiotic treatment, it should be remembered that such treatment inevitably leads to the occurrence of resistant strains that in the long term will make antibiotics ineffective. This suggests that vaccination, which classically is the most effective way to prevent and control infectious diseases in a large population, could be used to prevent infection and possibly also to treat the disease.
The increasing importance of H. pylori in the induction of a wide variety of gastric pathologies has represented a major challenge for the development of efficacious prophylactic and/or therapeutic strategies. To better understand the interactions between the bacterium and the host, much effort has focused on the development of appropriate animal models of infection reproducing aspects of the natural human infection.
Several animal models of infection and disease have been developed aiming at studying the pathogenesis of infection and development of preventive and therapeutic strategies. Many of these models are highly impractical, since they employ monkeys (Dubois, A., et al. 1994. Gastroenterology. 10:1405-1417) or species that are kept under gnotobiotic (that is, germ-free) conditions, for example germ-free dogs or piglets (Krakowka, S., et al. 1987. Infect. Immun. 55: 2789-2796; Radin, M. J., et al. 1990. Infect. Immun. 58: 2606-2612). Colonization of gnotobiotic piglets (Krakowka et al, 1987) has been reported using H. pylori strains isolated from patients with gastroduodenal diseases. However piglets cannot be kept under germ-free conditions for more than 2 months (Radin et al., 1990) mainly due to their nutritional needs.
Successful infection of specific pathogen-free (SPF) cats has been described using a strain isolated from conventional cats (Fox, J. G., et al. 1995. Infection and Immunity. 63: 2674-2681; Handt, L. K., et al. 1995. J. Clin. Microbiol. 33:2280-2289). Gnotobiotic beagle pups have also been infected with a human H. pylori isolate and kept under germ-free conditions for 30 days. However, in this model no data are available on long term infections with H. pylori (Radin et al., 1990). Other experimental animal models include athymic nu/nu or germ-free mice (Karita, M., et al. 1991. Am. J. Gastroenterol. 86:1596-1603).
The major drawbacks of these experimental infections, however, are the sophisticated and expensive housing systems required, and, more importantly, the peculiar immunological status of the gnotobiotic or immunodeficient hosts employed. More recently, H. pylori, freshly isolated from human gastroduodenal biopsies, have been adapted to persistently colonize the gastric mucosa of xenobiotic mice (Marchetti et al., 1995). This model has proven particularly useful to assess the feasibility of either preventive (Manetti, R., et al. 1997. Infect. Immun. 65:4615-4619; Marchetti et al., 1995; Marchetti, M., et al. 1998. Vaccine 16: 33-37; Radcliff, F. J., et al. 1997. Infect. Immun. 65:4668-4674) or therapeutic (Ghiara, P., et al. 1997. Infect. Immun. 65:4996-5002) vaccination, as well as for the in vivo screening of anti-H. pylori antimicrobials (Lee, A., et al. 1997. Gastroenterol. 112:1386-1397), and for studying the pathogenesis of infection (Sakagami, T., et al. 1996. Gut. 39:639-648). However, to evaluate gastric infection, mice have to be sacrificed; the pathological changes induced by the chronic infection and/or the effect of therapeutic or immunizing regimens cannot, therefore, be followed up in the same individual animal.
In United Kingdom patent application GB 9801000.2 (filed 16th Jan. 1998) and associated International patent application PCT/IB99/00217 (filed 15th Jan. 1999), there is described for the first time an animal model which can reproduce symptoms which have been clearly associated with the acute phases of infection with H. pylori in humans (Marshall, B. J., et al. 1985. Med. J. Australia. 142:436-439; Mitchell, J. D., et al. 1992. Am. J. Gastroenterol. 87:382-386; Morris, A., and G. Nicholson. 1987. Am. J. Gastroenterol. 82:192-199; Sobala, G. M., et al. 1991, Gut 32:1415-1418). The invention described in GB 9801000.2 and PCT/IB99/00217 is based on the discovery that H. pylori can persistently colonize the gastric mucosa of conventional xenobiotic dogs, and that this colonization causes acute symptoms, histopathological lesions and elicits specific immune responses. Thus, the animal model provided in GB 9801000.2 and PCT/IB99/00217 is ideal for studying the efficacy of treatments for H. pylori infection.
As H. pylori is a mucosa-related infection, where the bacteria do not invade the surrounding host cells, attempts to develop vaccines or treatments against the disease have concentrated on mucosal administration, specifically oral administration into the gastro-intestinal tract. Thus, as it had previously been thought that local (mucosal) treatment at the site of the H. pylori infection was necessary, the thrust of research in this area has been to develop mucosa-associated anti-H. pylori antibodies by the mucosal administration of prophylactics/therapeutics (see, for example: Chen, M., et al. 1992. Lancet 339:1120-1121; Ferrero, R. L., et al. 1994. Infect. Immun. 62:4981-4989; Michetti, P., et al. 1994. Gastroenterology 107:1002-1011; Lee, A., et al. 1994. Infect. Immun. 62:3594-3597; Doidge, C., et al. 1994. Lancet 343:974-979; Marchetti et al., 1995; Lee, C. K., et al. 1995. J. Infect. Dis. 172:161-172; Corthesy-Theulaz, I., et al. 1995. Gastroenterology 109:115-121; Cuenca, R., et al. 1996. Gastroenterology 110:1770-1775; Radcliff, F. J., et al. 1996. Vaccine 14:780-784; Stadtlander, C. T. K. H., et al. 1996. Dig. Dis. Sci. 41:1853-1862; Ferrero, R. L., et al. 1997. Gastroenterology 113:185-194; Weltzin, R., et al. 1997. Vaccine 15:370-376; Radcliff et al., 1997; Ghiara et al., 1997; Marchetti et al., 1998).
However, it has been shown in the present invention that a systemic protective effect against challenge with infectious H. pylori can be unexpectedly achieved using a non-mucosally administrated H. pylori antigen-containing composition. Specifically, it has been shown that, for instance, intramuscular (i.m.) immunization with whole H. pylori cell lysate can protect dogs against challenge with infectious H. pylori, and that the i.m. route, as an example of a non-mucosal route, can, unexpectedly, be considered for vaccination against this bacterium. Such a method may also be useful therapeutically in treating an already established H. pylori infection.
All documents (including patents, patent applications, research articles and books) which are mentioned in this application are hereby incorporated in their entirety by reference.